Single cell analysis by polymerase cycling assembly

ABSTRACT

The invention provides a method of making measurements on individual cells of a population, particularly cells that have identifying nucleic acid sequences, such as lymphoid cells, in one aspect, the invention provides a method of making multiparameter measurements on individual cells of such a population by carrying out a polymerase cycling assembly (PCA) reaction to link their identifying nucleic acid sequences to other cellular nucleic acids of interest. The fusion products of such PCA reaction are then sequenced and tabulated to generate multiparameter data for cells of the population.

This application is a continuation-in-part of co-pending U.S.application Ser. No. 12/615,263 filed 9 Nov. 2009, and claims priorityfrom co-pending U.S. provisional applications Ser. No. 61/332,175 filed6 May 2010, Ser. No. 61/446,822 filed 25 Feb. 2011, and Ser. No.61/452,594 filed 14 Mar. 2011, all of which are incorporated herein byreference in their entireties.

BACKGROUND

Cytometry plays an indispensable role is many medical and researchfields.

Image-based and flow cytometers have found widespread use in thesefields for counting cells and measuring their physical and molecularcharacteristics, e.g. Shapiro, Practical Flow Cytometry, 4th Edition(Wiley-Liss, 2003). In particular, flow cytometry is a powerfultechnique for rapidly measuring multiple parameters on large numbers ofindividual cells of a population enabling acquisition of statisticallyreliable information about the population and its subpopulations. Thetechnique has been important in the detection and management of a rangeof diseases, particularly blood-related diseases, such as hematopoieticcancers, HIV, and the like, e.g. Woijciech, Flow Cytometry in NeoplasticHematology, Second Edition (Informs Healthcare, 2010); Brown et al,Clinical Chemistry, 46: 8(B): 1221-1229 (2000). Despite this utility,flow cytometry has a number of drawbacks, including limited sensitivityin rare cell detection, e.g. Campana et al, Hematol. Oncol. Clin, NorthAm., 23(5); 1083-1098 (2009); limitations in the number of cellparameters that can be practically measured at the same time; and costlyinstrumentation.

In view of the above, it would be advantageous to many medical andresearch fields if there were available alternative methods and systemsfor making multiparameter measurements on large numbers of individualcells that overcame the drawbacks of current cytometric approaches.

SUMMARY OF THE INVENTION

The present invention is directed to methods for making multiparametermeasurements of target nucleic acids in single cells of a population,particularly cells such, as lymphocytes that contain cell-specificrecombined sequences. Aspects of the present invention are exemplifiedin a number of implementations and applications, some of which aresummarized below and throughout the specification.

In one aspect the invention includes a method of analyzing a pluralitytarget nucleic acids in each cell of a population comprising the stepsof: (a) providing multiple reactors each containing a single cell in a.polymerase cycling assembly (PCA) reaction mixture comprising a pair ofouter primers and one or more pairs of linking primers specific for theplurality of target nucleic acids; (b) performing a PCA reaction in thereactors to form fusion products of the target nucleic acids in thereactors; and (c) sequencing the fusion products from the reactors toidentity the target nucleic acids of each cell, in the population.

In another aspect the invention includes a method of distinguishingmultiple subpopulations of lymphocytes comprising the steps of: (a)providing multiple reactors each containing a single lymphocyte in apolymerase cycling assembly (PCA) reaction mixture comprising a pair ofouter primers and one or more pairs of linking primers, one or morepairs of such primers being specific for one or more target nucleicacids and at least one pair of such primers being specific for a nucleicacid containing a clonotype; (b) performing a PCA reaction in eachreactor to form a fusion product comprising the target nucleic acids anda clonotype of the lymphocyte therein; (c) sequencing the fusionproducts from the reactors; and (d) classifying each lymphocyte into asubpopulation by the target nucleic acids associated with its clonotype.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustrated,implementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the present invention.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 illustrates steps of one embodiment of the method of theinvention.

FIGS. 2A-2C illustrate a PCA scheme for linking target sequences wherepairs of internal primers have complementary tails.

FIGS. 3A-3C illustrate a PCA scheme for linking target sequences whereonly one primer of each pair of internal primers has a tail mat iscomplementary to an end of a target sequence.

FIGS. 4A-4C illustrate a PCA scheme for linking target sequences wherepairs of internal primers have complementary tails and external primershave tails for continued amplification of an assembled product by PCR.

FIGS. 5A-5F illustrate a multiplex of pairwise assemblies of targetsequences.

FIGS. 6A-6E illustrate a method of using PCA to link together threesequences.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, molecular biology (including recombinant techniques), cellbiology, and biochemistry, which are within the skill of the art. Suchconventional techniques include, but are not limited to, sampling andanalysis of blood cells, nucleic acid sequencing and analysis, and thelike. Specific illustrations of suitable techniques can be had byreference to the example herein below. However, other equivalentconventional procedures can, of course, also be used. Such conventionaltechniques and descriptions can be found in standard laboratory manualssuch as Genome Analysis: A laboratory Manual Series (Vols. HV); PCRPrimer; A Laboratory Manual; and Molecular Cloning: A Laboratory Manual(ail from Cold Spring Harbor Laboratory Press); and the like.

The invention provides a method of making measurements on individualcells of a population, particularly cells that have identifying nucleicacid sequences, such as lymphoid cells. In one aspect the inventionprovides a method of making multiparameter measurements on individualcells of such a population. An objective of assays of the invention, isto carry out a polymerase cycling assembly (PCA) reaction on individualcells to link their identifying nucleic acid sequences to other cellularnucleic acids of interest (referred to herein as “target nucleicacids”), the products of such, linking being referred to herein as“fusion products.” After their generation, fusion products can besequenced and tabulated to generate data, especially multiparameterdata, for each cell of a population. In one aspect, such data mayinclude gene expression data, data on the presence or absence of one ormore predetermined genomic sequences, gene copy number data, orcombinations of the foregoing. FIG. 1 gives an overview on oneembodiment of the invention. Lymphoid cells (100) each have a distinctidentifying nucleic acid (102), which in the figure is represented as aunique binary number. In one aspect, the identifying nucleic acids arethe clonotypes of the lymphocytes. In addition, each cell has and/orexpresses various nucleic acids of interest (104), or target nucleicacids, represented by the letters “a”, “b”, “c” and “w”, which may begenomic DNA, expressed genes, or the like. Cells (100) are disposed(106) in single cell reactors (110), which in this example areillustrated as micelles of a water-in-oil emulsion (108), although avariety of single cell reactors may be used, including but not limitedto, plates with arrays of nanoliter-volume wells, microfluidic devices,and the like, as described more fully below. In one aspect, single-cellemulsion (126) is generated using a microfluidic emulsion generator,such as disclosed by Zeng et al. Anal. Chem., 82: 3183-3190 (2010), orthe like.

Reactors (110) contain a PC A reaction mixture that, for example, maycomprise a nucleic acid polymerase, outer primers and linking primers(described more fully below), nucleoside triphosphates, a buffersolution, and the like. In some embodiments, a PCA reaction mixture mayalso include one or more cell lysing reagents, to access of suchreagents to target nucleic acids. For each reactor (110) containing acell, PCA reaction (112) generates fusion products (114) that maycomprise one or more pairs of sequences, such that one member of thepair is the identifying nucleic acid of the cell in the reactor and theother member is a nucleic acid of interest, such as an expressed gene.In other embodiments, fusion products may comprise triplets ofsequences, or higher order concatenations. In the method of theinvention, a single kind of fusion product may be generated for eachcell (or per reactor) or a plurality of different kinds of fusionproducts may be generated for each cell (or per reactor). Such pluralitymay be in the range of from 2 to 500, or from 2 to 200, or from 2 to100, or from 2 to 20. In one embodiment, such plurality may be in therange of from 2 to 10.

After completion of PCA reaction (112), emulsion (126) is broken andfusion products (114) are isolated (116). Fusion products (114) arerepresented in FIG. 1 as conjugates (118) of identifying nucleic acids(102) and target nucleic acids (128), A variety of conventional methodsmay be used to isolate fusion products (114), including, but not limitedto, column chromatography, ethanol precipitation, affinity purificationafter use of biotinylated primers, gel electrophoresis, or the like. Aspart of PCA reaction (112) or after isolation (116), additionalsequences may be added to fusion products (114) as necessary forsequencing (120), Sequencing may be carried out using a conventionalhigh-throughput instrument (122), e.g. Genome Analyzer IIx (Iliumina,Inc., San Diego), or the like. Data from instrument (122) may beorganized and displayed (124) in a variety of ways. In particular, wheretarget nucleic acids are selected gene expression products, e.g. mRNAs,plots maybe constructed that display per-cell expression levels ofselected gene for an entire population, or subpopulation, in a mannersimilar to that for flow cytometry data, as illustrated by plot (130).Each cell is associated with a unique clonotype that is linked via thePCA reaction to genes expressed in the cell in a proportion related totheir cellular abundance. Thus, by counting the number of expressed genesequences linked to a specific clonotype sequence, one obtains a measureof expression for such gene in the cell associated with the specificclonotype. As illustrated in plot (130), three subpopulations of cellsare indicated by the presence of separate clusters (132, 134, and 136)based on expression levels of gene w and gene a. In one aspect, whenevergene expression levels are monitored, at least one gene is selected asan internal standard for normalizing the expression measurements ofother genes.

Polymerase Cycling Assembly (PCA) Reaction Formats

Polymerase cycling assembly (PCA) reactions permit a plurality ofnucleic add fragments to be fused together to form a single fusionproduct in one or more cycles of fragment annealing and polymeraseextension, e.g. Xiong et al, FEBS Micro biol Rev., 32: 522-540 (2008).PCA reactions come in many formats. In one format of interest, PCAfollows a plurality of polymerase chain reactions (PCRs) taking place ina common reaction volume, wherein each component PCR includes at leastone linking primer that permits strands front the resulting amplicon toanneal to strands from another amplicon in the reaction and to heextended to form, a fusion product or a precursor of a fusion product.PCA in its various formats (and under various alternative names) is awell-known method for fragment assembly and gene synthesis, severalforms of which are disclosed in the following references: Yon et alNucleic Acids Research, 17: 4895 (1989); Chen et al, J. Am. Chem. Soc.,116: 8799-8800 (1994); Stemmer et al, Gene, 164: 49-53 (1995); Hoover etal, Nucleic Acids Research, 30; e43 (2002); Xiong et al, BiotechnologyAdvances, 26: 121-134 (2008); Xiong et al, FEBS Microbiol Rev, 32;522-540 (2008); and the like.

Some PCA formats useful in the present invention are described in FIGS.2A-2C, 3A-3C, 4A-4C, 5A-5D. and 6A-6E. FIGS. 2A-2C illustrate anexemplary PCA scheme (“Scheme 1”) for joining two separate fragments A′(208) and B′ (210) into a single fusion product (222). Fragment A′ (208)is amplified with primers (200) and (202) and fragment B′ (210) isamplified with primers (206) and (204) in the same PCR mixture. Primers(200) and (206) are “outer” primers of the PCA reaction and primers(202) and (204) are the “inner” primers of the PCA reaction. Innerprimers (202) and (204) each have a tail (203 and 205, respectively)that are not complementary to A′ or B′ (or adjacent sequences if A′ andB′ are segments imbedded in a longer sequence). Tails (203) and (205)are complementary to one another. Generally, such inner primer tails areselected for selective hybridization to its corresponding inner primer(and not elsewhere); but otherwise such tails may vary widely in lengthand sequence. In one aspect, such tails have a length in the range offrom 8 to 30 nucleotides; or a length in the range of from 14 to 24nucleotides. As the PCRs progress (212), product fragments A (215) and B(217) are produced that incorporate tails (203) and (205) into endregions (214) and (216), respectively. During the PCRs product fragmentsA (215) and B (217) will denature and some of the “upper”strands (215 a)of A anneal (218) to lower strands (217 b) of B and the 3′ ends areextended (219) to form (220) fusion product A-B (222). Fusion productA-B (222) may he further amplified by an excess of outer primers (200)and (206), In some embodiments, the region of fusion product (222)formed from, tails (203) and (205) may include one or more primerbinding sites for use in later analysis, such as high-throughputsequencing. Typically, in PDA reactions the concentrations of outerprimers are greater than the concentrations of inner primers so thatamplification of the fusion product continues alter initial formation.For example, in one embodiment for fusing two target nucleic acids outerprimer concentration may be from about 10 to 100 times that of the innerprimers, e.g. 1 μM for outer primers and 0.01 μM for inner primers.Otherwise, a PC A reaction may comprise the components of a PCR.

A variation of Scheme 1 is illustrated in FIGS. 3A-3C as Scheme 1(a). Asabove, fragment A (300) is amplified using primers (304) and (306) andfragment B′ (302) is amplified using primers (308) and (312) in PCRscarried out in a common reaction mixture. Outer primers (304) and (312)are employed as above, and inner primer (308) has tail (310); however,instead of tail (310) being complementary to a corresponding tail onprimer (306), it is complementary to a segment on the end of fragment A,namely, the same segment that primer (306) is complementary to. The PCRsproduce (315) fragments A and B, where 13 is identical to B′ (302) withthe addition of segment (316) created by tail (310) of primer (308). Asabove, as temperature cycling continues (particularly as inner primersbecome exhausted), the upper fragments of fragment A anneal (318) to thelower fragment of fragment B and are extended to produce fusion productA-B (320), which may be further amplified using primers (304) and (312).

Another embodiment of a PCA that may be used with the invention (“Scheme2”) is illustrated in FIGS. 4A-4C. The embodiment is similar to that ofFigs, 2A-2C, except that outer primers (404) and (414) have tails (408)and (418). respectively, which permit further amplification of a fusionproduct with predetermined primers. As discussed more fully below, thisembodiment is well-suited for multiplexed amplifications. Fragment A′(400) is amplified with primers (404) and (406), having tails (408) and(410), respectively, to produce fragment A, and fragment B′ (402) isamplified with primers (412) and (414), having tails (416) and (418),respectively, to produce (420) fragment B. Tails (410 and 416) of innerprimers (406 and 412) are selected to complementary (415) to oneanother. Ends of fragments A and B are augmented by segments (422, 424,426 and 428) generated by tails (408, 410, 416 and 418, respectively).As with previously described embodiments, upper strands of fragment Aanneal (430) to lower strands of fragment B and are extended (432) toform (434) fusion product A-B (436) that may be further amplified (437)using primers (438 and 440) that are the same as primers (404 and 414),but without tails.

As mentioned above, the embodiment of FIGS. 4A-4C, may be used in amultiplex PCA reaction, which is illustrated in FIGS. 5A-5D. Therefragments As (503), B′ (502), C (503), and D′ (504) are amplified, inPCR.s in a common reaction mixture using primer sets (506 and. 508) forfragment A′, (514 and 516) for fragment B′, (522 and 524) for C′ and(530 and 532) for D′. All primers have tails; outer primers (506, 516,522 and 532) each have tails (512, 520, 526 and 536, respectively) thatpermit both fragment amplification and subsequent fusion productamplification. Sequences of tails (512) and (520) may be the same ordifferent from the sequences of tails (526) and (536), respectively. Inone embodiment, the sequences of tails (512, 520, 526 and 536) are thesame. Tails of inner primers (518 and 510) are complementary (511) toone another; likewise, tails of inner primers (528 and 534) arecomplementary (513) to one another. The above PCRs generate fragments A(541), B (542), C (543) and D (544), which further anneal (546) to oneanother to form complexes (548 and 550) which are extended to formfusion products A-B (552) and C-D (554), respectively.

FIGS. 5E and 5F illustrate a generalization of the above embodiment inwhich multiple different target nucleic acids (560), A₁′, A₂′, . . .A_(g)′, are linked to the same target nucleic acid, X′ (562) to form(564) multiple fusion products X-A₁, X-A₂, . . . X-A_(k) (566). Thisembodiment is of particular interest when target nucleic acid, X, is asegment of recombined sequence of a lymphocyte, which can be used as atag for the lymphocyte that it originates from. In one aspect, X is aclonotype, such as a segment of a V(D)J region of either a B cell or Tcell. In one embodiment, a plurality of target nucleic acids. A₁, A₂, .. . A_(k), are fused to the clonotype of its cell of origin. In anotherembodiment, such plurality is between 2 and 1000; and in anotherembodiment, it is between 2 and 100; and in another embodiment, it isbetween 2 and 10. In PCA reactions of these embodiments, theconcentration of inner primer (568) may be greater than those of innerprimers of the various A,, nucleic acids so that there is adequatequantities of the X amplicon to anneal with the many stands of the A₁,amplicons in accordance with a method, of the invention, the fusionproducts (566) are extracted from the reaction mixture (e.g. viaconventional double stranded DNA purification techniques, such asavailable from Qiagen, or the like) and sequenced. The sequences of theouter primers may be selected to permit direct use for cluster formationwithout further manipulation for sequencing systems such as a GenomeAnalyzer (Illumina, San Diego, Calif.). In one aspect, X may be aclonotype and A₁, A₂, . . . A_(K) may be particular genes or transcriptsof interest. After sequencing fusion products, per cell gene expressionlevels may be tabulated and/or plotted as shown in FIG. 1.

In addition to multiplexed PCA reactions in a parallel sense tosimultaneously generate multiple binary fusion products, as illustratedin FIGS. 6A-6E, PCA reactions may be multiplexed in a serial sense toassemble multi-subunit fusion products. As shown in FIG. 6A, fragmentsA′ (601), B′ (602) and O (603) are amplified in a common PCR. mixturewith primer sets (606 and 608) for A′, (610 and 612) for B′ and (614 and616) for C′. All primers have tails; (i) tails (620 and 630) of outerprimers (606 and 616) are selected for amplification of outer fragmentsA′ and C′ and further amplification of three-way fusion product A-B-C(662) shown in FIG. 6E; (ii) tails (622 and 624) of inner primers (608and 610) are complementary to one another; and (iii) tails (628 and 626)of inner primers (654 and 612) are complementary to one another. ThePCRs generate (632) fragments A (641), B (642) and C (643), which in thereaction form (644) complexes (646 and 648) comprising segments LS1 andLS2, respectively, which in turn arc extended to form (650) fusionproducts A-B (652) and B-C (654). These fusion products are denaturedand some cross anneal (658) to one another by way of the common Bfragment (656) to form a complex which is extended (660) to form fusionproduct A-B-C (662). Exemplary, PCA reaction, conditions for the abovereaction may be as follows: 39.4 μL distilled water combined with 10 μLof 10× buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, and0.01% gelatin), 2 μL of a 10 mM solution of each of the dNTPs, 0.5 μL ofTaq polymerase (5 units/μL), 1 μL of each outer primer (from a 100 μMstock solution) and 10 μL of each inner primer (from a 0.1 μM stocksolution).

Single Cell Analysis

As mentioned above, in one aspect of the invention, cells from apopulation are disposed in reactors each containing a single cell. Thismay be accomplished by a variety of large-scale single-cell reactorplatforms known in the art, e.g. Clarke et al, U.S. patent publication2010/0255471; Mathies et al, U.S. patent publication 2010/0285975; Eddet al, U.S. patent publication 2010/0021984; Colston et al, U.S. patentpublication 2010/0173394; Love et al, international patent publicationWO2009/145925; Muraguchi et al U.S. patent publication 2009/0181859;Novak, et al, Angew. Chem. Int. Ed., 50: 390-395 (2011); and the like,which are incorporated herein by reference. In one aspect, cells aredisposed in wells of a microwell array where reactions, such as PCAreactions, take place; in another aspect, cells are disposed in micellesof a water-in-oil emulsion, where micelles serve as reactors. Micellereactors generated by microfluidics devices, e.g. Mathies et al (citedabove) or Edd et al (cited above), are of particular interest becauseuniform-sized micelles may be generated and cells encounter lower shearand stress than in bulk emulsification processes.

Cells of a sample may be suspended in a PCA reaction mixture prior todisposition into reactors. In one aspect, a PCA reaction mixture issubstantially the same as a PCR reaction mixture with inner at least onepair of inner primers and at least one pair of outer primers.Optionally, a PCA reaction mixture may comprise a lysing agent tofacilitate access of the PCA reagents to target nucleic acids ofisolated cells. Lysing conditions of a PCA reaction may vary widely andmay be based on the action of heat detergent, protease, alkaline, orcombinations of such factors. The following references provide guidancefor selection of single-cell lysing conditions where a polymerase-basedamplification, such as PCA, is employed; Throuhill et al, PrenatalDiagnosis, 21; 490-497 (2001); Kim et al Fertility and Sterility, 92;814-818 (2009); and the like. Exemplary lysis conditions for use withPCA reactions are as follows: 1) cells in H₂O at 96° C. for 15 min,followed by 15 min at 10° C.; 2) 200 mM KOH, 50 mM dithiotheitol, heatto 65° C. for 10 min; 3) for 4 μL protease-based lysis buffer: 1 μL of17 μM SDS combined with 3 μL of 125 μg/mL proteinase K, followed byincubation at 37° C. for 60 min, then 95° C. for 15 min (to inactivatethe proteinase K); 4) for 10 μL of a detergent-based lysis buffer: 2 μLH₂O, 2 μL 250 ng/μl, poly A, 2 μL 10 mM EDTA, 2 μL 250 mMdithiothreitol, 2 μL 0.5% N-laurylsarcosin salt solution. Single-cellanalysis platforms, incubation times, lysis buffer and/or PCA reactionother components, their concentrations, reactions volumes and the like,are design choices that are optimized for particular applications by oneof ordinary skill in the art.

Nucleic Acid Sequencing Techniques

Any high-throughput technique for sequencing nucleic acids can be usedin the method of the invention. DNA sequencing techniques includedideoxy sequencing reactions (Sanger method) using labeled terminatorsor primers and gel separation in slab or capillary, sequencing bysynthesis using reversibly terminated labeled nucleotides,pyrosequencing, 454 sequencing, sequencing by synthesis using allelespecific hybridization to a library of labeled clones that is followedby ligation, real time monitoring of the incorporation, of labelednucleotides during a polymerization step, polony sequencing, SOLIDsequencing, and the like. These sequencing approaches can thus be usedto sequence fusion products of target nucleic acids of interest andclonotypes based on T-cell receptors (TCRs) and/or B-cell receptors(BCRs). in one aspect of the invention, high-throughput methods ofsequencing are employed that comprise a step of spatially isolatingindividual molecules on a solid surface where they are sequenced inparallel. Such solid surfaces may include nonporous surfaces (such as inSolexa sequencing, e.g. BentSey et al, Nature 456: 53-59 (2008) orComplete Genomics sequencing, e.g. Drmanac et at Science, 327; 78-81(2010)), arrays of wells, which may include bead- or particle-boundtemplates (such as with 454, e.g. Margulies et al, Nature, 437: 376-380(2005) or Ion Torrent sequencing, U.S. patent publication 2010/0137143or 2010/0304982), micromachined membranes (such as with SMRT sequencing,e.g. Bid et al. Science, 323: 133-138 (2009)), or bead arrays (as withSOLID sequencing or polony sequencing, e.g. Kim et al, Science, 316:1481-1414 (2007)). In another aspect, such methods comprise amplifyingthe isolated molecules either before or after they are spatiallyisolated on a solid surface. Prior amplification may compriseemulsion-based amplification, such as emulsion PCR, or rolling circleamplification. Of particular interest is Solexa-based sequencing whereindividual template molecules arc spatially isolated on a solid surface,after which they are amplified in parallel by bridge PCR to formseparate clonal populations, or clusters, and then sequenced, asdescribed in BentSey et al (cited above) and in manufacturer'sinstructions (e.g. TraSeq™ Sample Preparation Kit and Data Sheet,Illumina, Inc., San Diego, Calif., 2010); and further in the followingreferences: U.S. Pat. Nos. 6,090,592; 6,300,070; 7,115,400; andEP0972081B1; which are incorporated by reference. In one embodiment,individual molecules disposed and amplified on a solid surface formclusters in a density of at least 10⁶ clusters per cm²; or in a densityof at least 5×10⁵ per cm²; or in a density of at least 10⁶ clusters percm². In one embodiment, sequencing chemistries are employed havingrelatively high error rates, in such embodiments, the average qualityscores produced by such chemistries are monotonically decliningfunctions of sequence read lengths. In one embodiment, such declinecorresponds to 0.5 percent of sequence reads have at least one error inpositions 1-75; 1 percent of sequence reads have at least one error inpositions 76-100; and 2 percent of sequence reads have at least oneerror in positions 101-125.

In one aspect of the invention, multiplex PCR is used to amplify membersof a mixture of nucleic acids, particularly mixtures comprisingrecombined immune molecules such as T cell receptors, B cell receptors,or portions thereof Guidance for carrying out multiplex PCRs of suchimmune molecules is found in the following references, which areincorporated by reference: Morley, U.S. Pat. No. 5,296,351; Gorski, U.S.Pat. No. 5,837,447; Dau, U.S. Pat. No. 6,087,096; Von Dongen et al, U.S.patent publication 2006/0234234; European patent publication EP1544308B1; Faham et al, U.S. patent publication 2010/0151471; Han, U.S.patent publication 2010/0021896; Robins et al, U.S. patent publication2010/033057; and the like. Such amplification techniques are readilymodified by those of ordinary skill in the art to supply outer primersand linking primers of the invention.

Cancer-Related Applications

Detecting cross-lineage rearrangements. Some types of otherwise uncommonrearrangements are common in some cancers and thus can be used toassociate them with tumor. For example, cross lineage rearrangements,like T cell receptor (α, β, γ and/or δ) in B cells or B cell receptor(IgH, IgK, and/or IgL) in T cells are common, especially in ALL. Thepresence of cross lineage rearrangements is likely to support amalignant origin of the clonotype. Demonstrating cross lineagerearrangement can be done by performing linked PCR on a cell by cellbasis. Linked PCR amplifies two distinct targets (for example IgH andTCRβ) and create a linked molecule between the two amplified targets.These targets from all the amplified cells can be then pooled andsequenced without losing the information as to whether the two targetsare expressed in the same or different cells. In order to getamplification even in the absence of the other rearrangement, anothercompeting product may be used. For example, for B cells, IgHamplification will always occur while the cross lineage TCRβ may or maynot occur. Two competing set of primers can be used for theamplification of TCRβ: one that amplifies the rearranged sequences andthe other the germ line sequences. Optionally, the two competing setscan he used at different concentrations allowing the rearranged sequenceto compete more efficiently when present in the cell. Ail the cellswould have their IgH and TCRβ products amplified and linked, andsequencing would be used to identify those cells with cross lineagerearrangement. Methods for achieving linked PCR are disclosed above. Onemethod to detect cells that do not have functional sequences uses thelinked PCR technique mentioned above. In this case, the linking has tooccur for the two alleles of the same target. For this purpose, 3 stagePCR can be performed. The first PCR of an immune cell genomicrearrangement is done from one cell with a set of primers (primer A andB) that allow the rearrangement of both alleles to be amplified. PrimersA and B are then removed (e.g. by dilution) and a portion of these PCRproducts can he re-amplified with a second set of primers (C and D)which also allow the same product to be amplified. Primers C and B canadditionally be designed, to include a sequence homology at their 5′termini that allows these 2 PCR products (from A/B and from C/D) toanneal to each other and extend to produce a linked, product. Afterremoving primers from this reaction, the two PCR products can be mixedand re-amplified by PCR using primers A and D. The result is a linkingof the two products, and in 50% of molecules they will carry bothalleles. Sequencing would identify high frequency linked non-functionalsequences. Specific high frequency non-functional sequences that areconsistently linked to a second non-functional sequence are indicativeof the potential cancer cell

In addition to serving as a marker of cells that have become cancerousIgH is often one of the two pathological translocation partners inlymphoid neoplasms. One example is the t(11:14) that puts the J segmentof IgH in close proximity to the cycline D1 (CCND1) gene resulting inits overexpression. This rearrangement which is referred to as BCL1-IgHoccurs in as many as 60-70% of mantle cell lymphoma as well as otherlymphoid neoplasms (e.g., 20% of multiple myeloma). Another example ist(14:18) that puts the J segment of IgH in close proximity to BCL2resulting in its over expression. This rearrangement occurs in up to 90%of follicular lymphoma and 20% of large B cell lymphoma. Theserearrangements are typically identified by cytogenetics. Southernblotting, or FISH. PCR has the potential to identify rearrangement atvery high sensitivity and specificity as shown by BCR-ABL for thedetection of Philadelphia chromosome. Different PCR techniques have beenused to the assessment of translocations relevant to lymphoma, with therecently introduced real time PCR (e.g. for BCL2-IgH) being probably themost advanced. There are a few features of BCL1-IgH and BCL2-IgH thatmake their detection less sensitive and specific, than that of BCR-ABL.First, in contrast to BCR-ABL, BCL1-IgH and BCL2-IgH do not generate afission protein, and there is no splicing event that generatespredictable molecular structure, instead the breakpoints may span alarge region. There are common breakpoints that allow the detection ofup to 88% of BCL2-IgH using a combination, of primers and ˜40% of theBCL1-IgH. This results in missing some patients that have thetranslocation. Second, these rearrangements may be present in normalindividuals that would never get cancer. For example, BLC2-IgHtranslocation has been found at the level, of ˜10⁻⁵ in a large fractionof the normal individuals with over ˜4% carrying BCL2-IgH at a frequencyof >1/25 K. The frequency of BCL2-IgH gets higher with increasing age.It is also hypothesized that different people may have distinct levelsof “background” translocation. Presumably the presence of thistranslocation in normal sample is due to the fact that tumorgenesis is amulti-step process and the BCL2-IgH is not sufficient for tumors toemerge. The presence of this low level background puts a limit on thesensitivity of detection.

Amplification of with a pool of the I primers complementary to all the Jsegments and primers complementary to the regions upstream of the BCL1or BCL2 translocation breakpoints can be sequenced. This can generate amethod for sensitive detection of these translocations and the cancercells they appear in. First, deep sequencing of individual isolatedmolecules (e.g. 100 K or i million, reads) can allow the detection ofthe appropriate sequences from a small number of cells in a backgroundof amplifications of other loci. In addition, the problem of thebackground translocations in normal individuals may ameliorate theproblem that real time PGR suffer from. There is evidence that, at leastin some cases, the background translocations are not clonal, but ratherappear repeatedly in the same patient. Using sequencing one candistinguish the different translocation events to obtain frequency ofthe independent translocation events. Since the breakpoint of differenttranslocations is likely to be distinct translocation events can bedistinguished from each other. Alternatively or additionally, a linkingPCR using the translocation with a B or T cell receptor gene can be doneto provide a unique barcode for the translocation. The linking can alsobe done statistically using a set of dilution samples as describedabove.

Similarly additional data -relating to the status of the cell containingthe cancer-related clonotype can be used to predict likelihood ofrecurrence. For example, the presence of certain markers (surface ornon-surface) can be an indication of the functional status of the celland hence the likelihood of recurrence. Sequencing before and after thecapture of cells with the relevant markers can determine the fraction ofcells with the cancer clonotype that carry the relevant markers.Similarly some markers relevant to the likelihood of recurrence (e.g.,expression of some gene relating to cell growth) can be assessed at theRNA level. This can be done by several methods including linking PCR asdescribed above. Finally, it is possible that the level of immunereceptor specific RNA in the tumor cell can have functional consequenceand association with the likelihood of recurrence. This level can beassessed by doing linking PCR between a control gene 1 that can link toeither the immune receptor rearrangement or control gene 2. The relativefraction of the two products can be indicative of the relative amount ofthe RNA in the cell. Another method involves comparing the RNA level tothe DNA level of the immune receptor rearrangement The frequency of thecancer-specific clonotype in the DNA identifies the relative level ofthe cancer-specific clonotype. The frequency of the same clonotype can.then be assessed from RNA, and the relative frequency m RNA and in DNAcan. be followed. A change in this relative frequency can be indicativeof a change in the likelihood of recurrence,

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand. scope of the present invention. The present invention is applicableto a variety of sensor implementations and other subject matter, inaddition to those discussed above.

Definitions

Unless otherwise specifically defined herein, terms and symbols ofnucleic acid

chemistry, biochemistry, genetics, and molecular biology used hereinfollow those of standard treatises and texts in the field, e.g. Rombergand Baker, DNA Replication, Second Edition (W.H. Freeman, New York,1992); Lehmnger, Biochemistry, Second Edition (Worth Publishers, NewYork, 1975); Strachan and Read, Human Molecular Genetics, Second Edition(Wiley-Liss, New York, 1999); Abbas et al. Cellular and MolecularImmunology, 6^(th) edition (Saunders, 2007).

“Amplicon” means the product of a polynucleotide amplification reaction;that is, a clonal population of polynucleotides, which may be singlestranded or double stranded, which are replicated from one or morestarting sequences. The one or more starting sequences may be one ormore copies of the same sequence, or they may be a mixture of differentsequences. Preferably, amplicons are formed by the amplification of asingle starting sequence. Amplicons may be produced by a variety ofamplification reactions whose products comprise replicates of the one ormore starting, or target, nucleic acids. In one aspect, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products, in one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et alU.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491(“NASBA”); Lizardi, U.S. Pat. No. 5,854,033; Aono et al, Japanese patentpubl. JP 4-262799 (rolling circle amplification); and the like. In oneaspect, amplicons of the invention are produced by PCRs. Anamplification reaction may be a “real-time” amplification if a detectionchemistry is available that permits a reaction product to be measured asthe amplification reaction progresses, e.g. “real-time PCR” describedbelow, or “real-time NASBA” as described in Leone et al, Nucleic AcidsResearch, 26: 2150-21.55 (1998), and like references. As used herein,the term “amplifying” means performing an amplification reaction. A“reaction mixture” means a solution containing all the necessaryreactants for performing a reaction, which may include, but not belimited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like.

“Oonotype” means a recombined nucleotide sequence of a T cell or B cellencoding a T cell receptor (TCR) or B cell receptor (BCR), or a portionthereof. In one aspect, a collection of all the distinct clonotypes of apopulation of lymphocytes of an. individual is a repertoire of suchpopulation, e.g. Arstila et al. Science, 286: 958-961 (1999); Yassai etal, Immuoogenetics, 61: 493-502 (2009); Kedzierska et al, Mol. Immunol.,45(3); 607-618 (2008); and the like. A “clonotype profile,” or“repertoire profile,” is a tabulation or representation of clonotypes ofa population of T cells and/or B cells (such as a peripheral bloodsample containing such, cells) that includes substantially all of therepertoire's clonotypes and their relative abundances. As used herein,“clonotype profile,” “repertoire profile,” and “repertoire” are usedinterchangeably. (That is, the term “repertoire” as discussed more fullybelow, means a. repertoire measured from a sample of lymphocytes). Inone aspect of the invention, clonotypes comprise portions of animmunoglobulin heavy chain (IgH) or a TCR ft chain. In other aspects ofthe invention, may be based on other recombined molecules, such asimmunoglobulin light chains or TCRα chains, or portions thereof.

“Complementarity determining regions” (CDRs) mean regions of animmunoglobulin (i.e., antibody) or T cell receptor where the moleculecomplements an antigen's conformation, thereby determining themolecule's specificity and contact with a specific antigen. T cellreceptors and immunoglobulins each have three CDRs: CDR1 and CDR2 arefound in the variable (V) domain, and CDR3 includes some of V, all ofdiverse (D) (heavy chains only) and joint Q), and some of the constant(C) domains.

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the contest of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., primers,enzymes, etc, in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains primers.

“Micrafluidics device” means an integrated system of one or morechambers, ports, and channels that are interconnected and in fluidcommunication and designed for carrying out an analytical reaction orprocess, either alone or in cooperation with an appliance or instrumentthat provides support functions, such as sample introduction, fluidand/or reagent driving means, temperature control, detection systems,data collection and/or integration systems, and the like, Microfluidicsdevices may further include valves, pumps, and specialized functionalcoatings on interior walls, e.g. to prevent adsorption of samplecomponents or reactants, facilitate reagent movement by electroosmosis,or the like. Such devices are usually fabricated in or as a solidsubstrate, which may be glass, plastic, or other solid polymericmaterials, and typically have a planar format for ease of detecting andmonitoring sample and reagent movement, especially via optical orelectrochemical methods. Features of a microfluidic device usually havecross-sectional dimensions of less than a few hundred square micrometersand passages typically have capillary dimensions, e.g. having maximalcross-sectional dimensions of from about 500 μm to about 0.1 μm.Microfluidics devices typically have volume capacities in the range offrom 1 μL to a few nL, e.g. 10-100 nL. The fabrication and operation ofmicrofluidics devices are well-known in the art as exemplified by thefollowing references that, are incorporated by reference: Ramsey, U.S.Pat. Nos. 6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al,U.S. Pat. Nos. 5,1.26,022 and 6,054,034: Nelson et al, U.S. Pat. No.6,613,525; Maher et al, U.S. Pat. No. 6,399,952; Ricco et atInternational patent publication. WO 02/24322; Bjornson et al,International patent publication WO 99/19717; Wilding et al, U.S. Pat.Nos. 5,587,128; 5,498,392; Sia et al, Electrophoresis, 24: 3563-3576(2003); linger et al Science, 288: 113-116 (2000); Enzelberger et al,U.S. Pat. No. 6,960,437.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension, of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references; McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical.Approach (TRL Press, Oxford, 1991 and 1995, respectively). For example,in a conventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C.. The term “PCR” encompassesderivative forms of the reaction, including but not limited to, RT-PCR,real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and thelike. PCR reaction volumes typically range from a few hundrednanoliters, e.g. 200 nL, to a few hundred μL, e.g. 200 μL. “Reversetranscription PCR,” or “RT-PCR,” means a PCR that is preceded by areverse transcription reaction mat converts a target RNA to acomplementary single stranded DNA, which is then amplified, e.g. Tecottet al, U.S. Pat. No. 5,168,038, which patent is incorporated herein byreference. “Real-time PCR” means a PCR for which the amount of reactionproduct, i.e. amplicon., is monitored as the reaction proceeds. Thereare many forms of real-time PCR that differ mainly in the detectionchemistries used for monitoring the reaction product, e.g. Gelfand etal, U.S. Pat. No. 5,210,015 (“taqman”); Wittwer et al, U.S. Pat. Nos.6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al, U.S. Pat. No.5,925,517 (molecular beacons); which patents are incorporated herein byreference. Detection chemistries for real-time PCR are reviewed inMaekay et al. Nucleic Acids Research, 30: 1292-1305 (2002), which isalso incorporated herein by reference. “Nested PCR” means a two-stagePCR wherein the amplicon of a first PCR. becomes the sample for a.second PCR using a new set of primers, at least one of which binds to aninterior location of the first amplicon. As used herein, “initialprimers” in reference to a nested amplification reaction, mean theprimers used to generate a first amplicon, and “secondary primers” meanthe one or more primers used to generate a second, or nested, amplicon.“Multiplexed PCR” means a PCR wherein multiple target sequences (or asingle target sequence and one or more reference sequences) aresimultaneously carried out in the same reaction mixture, e.g. Bernard etal. Anal. Biochem., 273; 221-228 (1999) (two-color real-time PCR).Usually, distinct sets of primers are employed for each sequence beingamplified. “Quantitative PCR” means a PCR designed to measure theabundance of one or more specific target sequences in a sample orspecimen. Quantitative PCR includes both absolute quantitation andrelative quantitation of such target sequences. Quantitativemeasurements are made using one or more reference sequences that may beassayed separately or together with a target sequence. The referencesequence may be endogenous or exogenous to a sample or specimen, and inthe latter case, may comprise one or more competitor templates. Typicalendogenous reference sequences include segments of transcripts of thefollowing genes: β-actin, GAPDH, β₂-microglobulin, ribosomal RNA, andthe like. Techniques for quantitative PCR are well-known to those ofordinary skill in the art, as exemplified in the following referencesthat are incorporated by reference: Freeman et al, Biotechniques, 26:112-126 (1999); Becker-Andre et al, Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al, Biotechniques, 21: 268-279 (1996);Diviacco et al, Gene, 122: 3013-3020 (1992); Becker-Andre et al NucleicAcids Research, 17: 9437-9446 (1989); and the like.

“Polymerase cycling assembly” or “PCA” reaction (also referred to hereinas “linked PCR”) means a PCR that comprises at least one pair of outerprimers and at least one pair of inner primers. An inner primer has a 3′portion that is complementary to a target nucleic acid (or itscomplement) and a 5′ portion that is complementary to the 5′ portion ofanother inner primer corresponding to a different target nucleic acid,

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3″ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference; Dieffenbach,editor, PCR Primer; A Laboratory Manual, 2^(nd) Edition (Cold SpringHarbor Press, New York, 2003).

“Repertoire” means a set of distinct recombined. nucleotide sequencesthat encode T cell receptors (TCRs) or B cell receptors (BCRs), orfragments thereof respectively, in a population of lymphocytes of anindividual, wherein the nucleotide sequences of the set have aone-to-one correspondence with distinct lymphocytes or their clonalsubpopulations for substantially all of the lymphocytes of thepopulation. Member nucleotide sequences of a repertoire are referred toherein, as a “clonotype.” In one aspect, a repertoire comprises anysegment of nucleic acid, common to a T cell or a B cell population whichhas undergone somatic recombination during the development of TCRs orBCRs, including normal or aberrant (e.g. associated with cancers)precursors thereof, including, but not limited to, any of the following;an immunoglobulin heavy chain (IgH) or subsets thereof (e.g., an IgHvariable region, CDR3 region, or the like), an immunoglobulin lightchain or subsets thereof (e.g. a variable region, CDR region, or thelike), T cell receptor α chain or subsets thereof, T cell receptor βchain or subsets thereof (e.g. variable region, CDR3, V(D)J region, orthe like), a CDR (including CDR1, CDR2 or CDR3, of either TCRs or BCRs,or combinations of such CDRs), V(D)J regions of either TCRs or BCRs,hypermutated regions of IgH variable regions, or the like, in oneaspect, a repertoire is selected so that its diversity (i.e. the numberof distinct nucleic acid sequences in the set) is large enough so thatsubstantially every T cell or B cell or clone thereof in an individualcarries a unique nucleic acid sequence of such repertoire. That is, inaccordance with the invention, a practitioner may select for definingclonotypes a particular segment or region of recombined nucleic acidsthat encode TCRs or BCRs that do not reflect the full diversity of apopulation of T cells or B cells; however, preferably, clonotypes aredefined so that they do reflect the diversity of the population of Tcells and/or B cells from which they are derived. That is, preferablyeach different clone of a sample has different clonotype. In otheraspects of the invention, the population of lymphocytes corresponding toa repertoire may be circulating B cells, or may be circulating T cells,or may be subpopulations of either of the foregoing populations,including but not limited to, CD4+ T cells, or CD8+ T cells, or othersubpopulations defined, by cell surface markers, or the like. In oneembodiment, a repertoire of human TCR β chains comprises a number ofdistinct nucleotide sequences in the range of from 0.3×10⁶ to 1.8×10⁶,or in the range of from 0.5×10⁶ to 1.5×10⁶, or in the range of from0.8×10⁶ to 1.×10⁶. Such subpopulations may be acquired by taking samplesfrom particular tissues, e.g. bone marrow, or lymph nodes, or the like,or by sorting or enriching cells from a sample (such as peripheralblood) based on one or more cell surface markers, size, morphology, orthe like. In still other aspects, the population of lymphocytescorresponding to a repertoire may be derived from disease tissues, suchas a tumor tissue, an infected tissue, or the like. In a particularembodiment, a repertoire of the invention comprises a set of nucleotidesequences encoding substantially ail segments of the V(D)J region of anIgH chain. In one aspect, “substantially all” as used herein means everysegment having a relative abundance of 0.001 percent or higher; or inanother aspect, a relative abundance of 0.0001 percent or higher. Inanother particular embodiment, a repertoire of the invention comprises aset of nucleotide sequences that, encodes substantially all segments ofthe V(D)J region of a TCR β chain. In another embodiment, a repertoireof the invention comprises a set of nucleotide sequences having lengthsin the range of from 25-200 nucleotides and including segments of the V,D, and J regions of a TCR β chain. In another embodiment, a repertoireof the invention comprises a. set of nucleotide sequences having lengthsin the range of from 25-200 nucleotides and including segments of the V,D, and J regions of an IgH chain, in another embodiment, a repertoire ofthe invention comprises a number of distinct nucleotide sequences thatis substantially equivalent to the number of lymphocytes expressing adistinct IgH chain, in another embodiment, a repertoire of the inventioncomprises a number of distinct nucleotide sequences that issubstantially equivalent to the number of lymphocytes expressing adistinct TCR β chain. In still another embodiment, “substantiallyequivalent” means that with ninety-nine percent probability a repertoireof nucleotide sequences will include a nucleotide sequence encoding anIgH or TCR β or portion thereof carried or expressed by every lymphocyteof a population of an individual at a frequency of 0.001 percent orgreater. In still another embodiment, “substantially equivalent” meansthat with ninety-nine percent probability a repertoire of nucleotidesequences will include a nucleotide sequence encoding an IgH or TCR β orportion thereof carried or expressed by every lymphocyte present at afrequency of 0.0001 percent or greater. The foregoing sets of clonotypesare sometimes referred to herein as representing the “mil repertoire” ofIgH and/or TCRβ sequences.

What is claimed is:
 1. A method of analyzing a plurality target nucleicacids in each cell of a population, the method comprising the steps of:providing multiple reactors each containing a single cell in apolymerase cycling assembly (PCA) reaction mixture comprising a pair ofouter primers and one or more pairs of linking primers specific for tireplurality of target nucleic acids; performing a PCA reaction in thereactors to form, fusion products of the target nucleic acids in thereactors; and sequencing the fusion products from the reactors toidentify the target nucleic acids of each cell in the population.
 2. Themethod of claim 1 wherein said multiple reactors are aqueous micelles ofa water-in-oil emulsion,
 3. The method of claim 3 wherein saidwater-in-oil emulsion is generated by a microfluidics device.
 4. Themethod of claim 1 wherein said population is a population of B cellsand/or T cells.
 5. The method of claim 4 wherein at least one pair ofprimers from said, outer primers and said linking primers is specificfor a clonotype of said B cells and/or T cells.
 6. The method of claim 5wherein at least one pair of primers from said outer primers and saidlinking primers is specific for a nucleic acid sequence of said B cellsand/or T cells that is a cancer marker or encodes a cancer marker. 7.The method of claim 6 wherein said nucleic acid is an RNA that,indicates a cancerous state by over expression.
 8. The method of claim 6wherein said nucleic acid is a DNA that indicates a cancerous state byexcess copy number.
 9. A method of distinguishing multiplesubpopulations of lymphocytes, the method comprising the steps of:providing multiple reactors each containing a single lymphocyte in apolymerase cycling assembly (PCA) reaction mixture comprising a pair ofouter primers and one or more pairs of linking primers, at least onepair of such primers being specific for a nucleic acid containing aclonotype and one or more pairs of such primers being specific for oneor more target nucleic acids characteristic of the multiplesubpopulations of lymphocytes; performing a PCA reaction in each reactorto form a fusion product comprising the target nucleic acids and aclonotype of the lymphocyte therein; sequencing the fusion products fromthe reactors; and classifying each lymphocyte into a subpopulatioa bythe target nucleic acids associated with its clonotype.
 10. The methodof claim 9 wherein said multiple reactors are aqueous micelles of awater-in-oil emulsion.
 11. The method of claim 10 wherein saidwater-in-oil emulsion is generated by a microfluidics device.
 12. Themethod of claim 9 wherein said nucleic acid containing said clonotypeand said one or more target nucleic acids are RNA and wherein said stepof classifying includes determining the relative expression levels ofsaid one or more target nucleic acids.
 13. A method of detectingcross-lineage rearrangements in a population, of lymphocytes, the methodcomprising the steps of: providing multiple reactors each containing asingle lymphocyte in a polymerase cycling assembly (PCA) reactionmixture comprising a pair of outer primers and one or more pairs oflinking primers, at least one pair of such primers being specific for anucleic acid containing at least a portion of a B cell receptor gene andat least one pair of such primers being specific for a nucleic acidcontaining at least a portion of a T cell receptor gene; performing aPCA reaction in each reactor to form a fusion product comprising thetarget nucleic acids and a clonotype of the lymphocyte therein;sequencing the fusion products from the reactors; and determining thepresence; absence or level of fusion products that comprise both aportion of a B cell receptor gene and a portion of a T cell receptorgene to detect cross-lineage rearrangements in the population oflymphocytes.
 14. The method of claim 13 wherein said multiple reactorsare aqueous micelles of a water-in-oil emulsion.
 15. The method, ofclaim 14 wherein said water-in-oil emulsion is generated, by amicrofluidics device.